title: A multiplex technology platform for the rapid analysis of clinically actionable genetic alterations and validation for BRAF p.V600E detection in 1549 cytologic and histologic specimens.
abstract: BACKGROUND
  Current clinicopathologic assessment of malignant neoplastic diseases entails the analysis of specific genetic alterations that provide diagnostic, prognostic, or therapy-determining information.
  
  
  OBJECTIVE
  To develop and validate a robust molecular method to detect clinically relevant mutations in various tissue types and anatomic pathology specimens.
  
  
  METHODS
  Genes of interest were amplified by multiplex polymerase chain reaction and sequence variants identified by liquid bead array cytometry. The BRAF assay was fully characterized by using plasmids and genomic DNA extracted from cell lines, metastatic colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissues, and thyroid nodule fine-needle aspirates.
  
  
  RESULTS
  Qualitative multiplex assays for 22 different mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were established. The high signal-to-noise ratio of the technology enabled reproducible detection of BRAF c.1799T>A (p.V600E) at 0.5% mutant allele in 20 ng of genomic DNA. Precision studies with multiple operators and instruments showed very high repeatability and reproducibility with 100% (98.7%-100%) qualitative agreement among 292 individual measures in 38 runs. Evaluation of 1549 representative pathologic specimens in 2 laboratories relative to independent reference methods resulted in 99.0% (97.6%-99.6%) agreement for colorectal FFPE tissues (n = 416) and 98.9% (98.2%-99.4%) for thyroid fine-needle aspiration specimens (n = 1133) with an overall diagnostic odds ratio of 10 856 (2451-48 078).
  
  
  CONCLUSIONS
  The multiplex assay system is a sensitive and reliable method to detect BRAF c.1799T>A mutation in colorectal and thyroid lesions. This optimized technology platform is suitable for the rapid analysis of clinically actionable genetic alterations in cytologic and histologic specimens.
authors.affiliation: From Asuragen Inc, Austin, Texas, (Drs Smith, Beaudenon-Huibregtse, Laosinchai-Wolf, and Labourier); the Department of Pathology, Rouen University Hospital, Rouen, France (Drs Lamy and Sabourin); and INSERM U1079, Faculty of Medicine, Rouen University, Rouen, France (Dr Sesboüé).
authors.forename: David L
authors.initials: DL
authors.lastname: Smith
authors.forename: Aude
authors.initials: A
authors.lastname: Lamy
authors.forename: Sylvie
authors.initials: S
authors.lastname: Beaudenon-Huibregtse
authors.forename: Richard
authors.initials: R
authors.lastname: Sesboüé
authors.forename: Walairat
authors.initials: W
authors.lastname: Laosinchai-Wolf
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Sabourin
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Labourier
mesh_terms: D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D016606:Thyroid Nodule / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf